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WO2012012051A2 - Méthode de codétection multicolore de micro-arn et de protéines - Google Patents

Méthode de codétection multicolore de micro-arn et de protéines Download PDF

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WO2012012051A2
WO2012012051A2 PCT/US2011/040624 US2011040624W WO2012012051A2 WO 2012012051 A2 WO2012012051 A2 WO 2012012051A2 US 2011040624 W US2011040624 W US 2011040624W WO 2012012051 A2 WO2012012051 A2 WO 2012012051A2
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mir
expression
protein
mirna
cancer
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WO2012012051A3 (fr
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Lorenzo F. Sempere
Murray Korc
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Dartmouth College
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Dartmouth College
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Priority to US13/172,047 priority patent/US8580513B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • MicroRNAs are a class of short non-coding regulatory RNA genes, which act as post -transcriptional regulators of gene expression (Lee & Ambros (2001) Science 294 (5543) : 862-4 ; Lau, et al . (2001) Science 294 ( 5543 ) : 858 - 62; Lagos -Quintana, et al . (2001) Science 294 (5543) : 853-8) .
  • the -21-23 nucleotide-long miRNAs can trigger translat ional downregulation and/or increased degradation of mRNA of target genes (Bartel (2009) Cell 136 (2) : 215-33) .
  • the recent explosion of miRNA research in biomedical sciences and particularly in cancer biology attests to their importance to human disease (Ventura & Jacks (2009) Cell 136(4) :586- 91; Sempere & Kauppinen (2009) In: Handbook of Cell Signaling . 2 nd ed. Oxford: Academic Press, Bradshaw & Dennis (eds.) pg . 2965-81) .
  • RNA extracted from whole tissue biopsies has provided short lists of miRNAs that could serve as useful biomarkers for early detection, diagnosis and/or prognosis of different types of cancer (Barbarotto, et al . (2 ⁇ 08) Jnt. J. Cancer 122 (5) :969-77) .
  • Low levels of let-7, miR-34, miR- 126, miR-145 and high levels of miR-21, miR-155, miR-221 have been frequently reported in association with breast, colorectal, gastrointestinal, lung, pancreas, prostate and/or thyroid cancer (Barbarotto, et al . (2008) supra; Volinia, et al . (2006) Proc . Natl. Acad.
  • Locked nucleic acids a class of bicyclical high-affinity RNA analogues (Kauppinen, et al . (2006) Handb. Exp. Pharmacol. 173:405-22) have been shown to provide specific and avid hybridization to the short RNA sequence of mature miRNAs in zebrafish and mouse embryos by whole-mount ISH using chromogenic staining (Kloosterman, et al . (2006) Nat. Methods 3(l) :27-9; ienholds, et al . (2005) Science 309 (5732) : 310-1) .
  • ISH methods have been implemented to detect miRNA expression in formalin- fixed paraffin-embedded (FFPE) brain (Nelson, et al . (2006) RNA 12 (2) : 187-91) , breast (Sempere, et al . (2007) Cancer Res. 67 (24) : 11612-20) , colon (Yamamichi, et al . (2009) Clin. Cancer Res. 15 (12) : 4009-16) , lung (Liu, et al . (2010) J. Clin. Invest. 120 (4) : 1298-309) , and pancreatic tissue sections (Dillhoff, et al . (2008) J " . Gastrointest . Surg.
  • FFPE formalin- fixed paraffin-embedded
  • Protocols 5:1061-1073; US 2009/0156428) these methods do not contemplate removing or destroying probes or labels between the detection of the multiple targets. Therefore, interference from the reagents used in previous detection steps can decrease the sensitivity of these methods and limit the number of independent markers that can be codetected.
  • the present invention features methods and a kit for multicolor codetection of miRNA and proteins for use in diagnostic and prognostic application.
  • the method of the invention involves the steps of (a) contacting a biological sample with a probe that binds to a miRNA, (b) detecting binding of the probe to the miRNA with a label, (c) inactivating the label, (d) contacting the biological sample with a binding agent that specifically binds to a protein, and (e) detecting binding of the binding agent to the protein, thereby codetecting the miRNA and protein in the biological sample.
  • the label is composed of at least one hapten, an anti- hapten antibody, and an enzyme-conjugated secondary antibody specific for the anti-hapten antibody.
  • the enzyme of the enzyme- conjugated secondary antibody is horseradish peroxidase.
  • the probe is selected from the group of SEQ ID NO : 1 to 56 and is a modified probe.
  • the binding agent that specifically binds to the protein comprises an antibody and binding of the binding agent to the protein is detected by (i) contacting the antibody with an enzyme-conjugated secondary antibody, and (ii) detecting the enzyme activity, e.g., horseradish peroxidase activity.
  • the biological sample is a biopsy sample and the protein is selected from the group of amylase, cytokeratin (CK) 5/6, CK7, CK8/18, CK14, CK19, CK20; cluster of differentiation
  • CD 3, CD4, CD8, CDllb, CDllc, CD19, CD20, CD31, CD34, CD24, CD44, CD45, CD68, CD86, CD105; myeloperoxidase; E- cadherin; laminin; estrogen receptor (ER) ; Glucagon; Human Epidermal growth factor Receptor 2 (HER2); Insulin; Ki-67; phosphorylated v-akt murine thymoma viral oncogene (pAKT) ; protein 15 (pl5) , pl6, p21, p27, p53 , p63; mutS homolog 6
  • MSH-6 Proliferating Cell Nuclear Antigen (PCNA) ; Progesterone Receptor (PR) ; Phosphatase and Tensin Homolog
  • the method of the invention involves the steps of (a) contacting a biological sample with a probe that binds to a miRNA, (b) detecting binding of the probe to the miRNA with a label, (c) inactivating the label, (d) contacting the biological sample with an antibody that specifically binds to a protein, (e) detecting binding of the antibody to the protein with a secondary antibody reagent, (f) inactivating the secondary antibody reagent, and (g) repeating steps (a) to (f) for the codetection of multiple miRNAs and proteins in the biological sample .
  • a kit is composed of a probe that binds to a miRNA, and a binding agent that specifically binds to a protein, wherein the labeled probe is selected from the group of SEQ ID NO:l to 56.
  • Figure 1 shows the reproducibility of the ISH method and signal analysis.
  • Matched normal and tumor FFPE breast tissue sections were used to codetect miR-205 and U6 snRNA using FAM2X and Bio2X-tagged probes, respectively.
  • miR-205 and U6 signal was revealed by sequential TSA reactions with FITC- tyramine (green for miR-205 probe) and Rhodamine- tyramine (red for U6 probe) substrates.
  • Consecutive (i, ii, iii) matched normal (N) and tumor (T) tissue sections were assayed on separate days (1 and 2) .
  • Figure 1A shows the signal intensity of miR-205 measured across the mammary duct in Nl tissue and across invasive carcinoma lesion in Tl (upper panel) .
  • Line profile of Nliii was slightly shifted to the right (gap) to match the signal from myoepithelial (myo) and luminal (lu) cellular structures of Nli and Nlii.
  • Distribution of pixel intensity of the whole images indicates concordant readings for each intra- and inter-experimental replica (mid panel) .
  • Bar graph displays means and standard deviations of percentage of pixels within each intensity class for each set of intra -experimental replica (lower panel) .
  • Raw images of miR-205 and snRNA U6 were captured with the same exposure and gain setting in normal and tumor tissue were displayed as a heat map graph.
  • Figure IB shows the signal intensity of miR-205 and U6 expression. Background intensity was subtracted from the recorded intensity value and these corrected intensity values were used to generate line graphs.
  • Average intensity of miR-205 and U6 signal of selected structures was used to calculate relative fold decrease of miR-205 expression in luminal and cancer cells compared to myoepithelial cells (lu/myo and ICa/myo, respectively) .
  • Figure 2 shows the codetection of miRNAs with cell- type specific and prognostic protein markers.
  • Serial FFPE tissue sections of the indicated organs were stained with H&E to reveal histological features or subject to ISH assay using FAM2X-tagged probes against miR-34a, miR-126, miR- 141, miR-214 and miR-375 mixed with Bio2X-tagged probe against 18S rRNA, BrdU2X-tagged probe against miR-205 or DIG2X- tagged probe against let -7a.
  • miRNA signal was revealed by sequential TSA reactions with FITC-tyramine, Rhodamine- tyramine and A CA-tyramine substrates.
  • HIER heat-induced epitope retrieval
  • expression of indicated proteins was revealed by sequential TSA reactions with DYLIGHT594 -tyramine or DYLIGHT680-tyramine and AMCA- tyramine substrates, each TSA reaction was preceded by incubation with specific antibodies against each protein and appropriate anti-source species/HRP antibody; except for insulin expression, which was revealed by an anti -guinea pig secondary antibody conjugated to CY3 obviating the need for the TSA step.
  • Line profile analysis was used to quantitate intensity of RNA or protein expression. Background intensity was subtracted and intensity values were normalized setting the point with maximum intensity to 100 and calculating other values in relation to this reference. miRNA expression pattern was plotted as a line; independently, expression patterns of each protein were plotted as stacked areas .
  • FIG. 3 shows the co-localization of miR-155 with CD45 immune cell marker.
  • Serial 4- ⁇ FFPE tumor tissue sections of the indicated organs were stained with H&E to reveal histological features or subject to standard ISH assay using FA 2X-tagged probe against miR-155.
  • MiR-155 signal was revealed by TSA reaction with FITC-tyramine .
  • CK19 expression was revealed by TSA reaction with DYLIGHT680 -tyramine .
  • CD45 expression was revealed by TSA reaction with Rhodamine- tyramine .
  • Tissue sections were counterstained with nuclear marker DAPI .
  • Line profile analysis was used to quantify intensity of RNA or protein expression. Background intensity was subtracted and intensity values were normalized setting the point with maximum intensity to 100 and calculating other values in relation to this reference. miRNA expression pattern was plotted as a line; independently, expression patterns of each protein were plotted as stacked areas.
  • FIG. 4 shows the codetection of miR-21 with PTEN in clinical specimens.
  • Serial 4- ⁇ FFPE tumor tissue sections of the indicated organs were stained with H&E to reveal histological features or subject to standard ISH assay using FAM2X-tagged probe against miR-21 and Bio2X- tagged probe against snRNA U6.
  • MiR-21 and snRNA U6 signal was revealed by sequential TSA reactions with FITC-tyramine and AMCA-tyramine substrates.
  • Smooth muscle actin (SMA) expression was revealed by TSA reaction with DYLIGHT594.
  • VIM Vimentin
  • FIG. 5 is a schematic representation of an illustrative multi-color ISH/IHC assay.
  • a FFPE tumor tissue section is subject to standard ISH assay using FAM2X- tagged probe against a miRNA and Bio2X-tagged probe against snRNA U6.
  • the miRNA and snRNA U6 signal is revealed by sequential TSA reactions with FITC-tyramine (color 1) and AMCA- tyramine (color 2) substrates.
  • a protein marker such epithelial-specific cytokeratin 19 (CK19) is revealed by TSA reaction with DYLIGHT680 (color 3) .
  • DYLIGHT680 color 3
  • DYLIGHT680 DYLIGHT680
  • DYLIGHT680 DYLIGHT680
  • Expression of immune cell-specific (CD45) and mesenchymal cell-specific (SMA) marker is revealed by TSA reaction with Rhodamine-tyramine (color 4) and DYLIGHT594 (color
  • FFPE formalin- fixed paraffin embedded
  • the instant analysis describes a combination of ISH and IHC assays to study a subset of cancer- associated miRNAs, including frequently down-regulated ⁇ e.g., miR-34a and miR-126) and up-regulated (e.g., miR-21, miR-155 and miR-lOb) miRNAs, in a variety of cancers including breast, colorectal, lung, pancreas and prostate carcinomas.
  • up-regulated miR-21, miR-155 and miR-lOb miRNAs e.g., miR-21, miR-155 and miR-lOb miRNAs
  • ISH in combination with IHC for the detection of miRNAs and clinically important protein markers in FFPE specimens now provides a fluorescence-based multi-color ISH/IHC assay that is rapid, sensitive, compatible with current automated clinical IHC assays, and provides spatial characterization of miRNA expression .
  • the present invention features a method for the codetection of miRNA and protein in a biological sample by (a) contacting a biological sample with a probe that binds to a miRNA, (b) detecting binding of the probe to the miRNA with a label, (c) inactivating the label, (d) contacting the biological sample with a binding agent that specifically binds to a protein, and (e) detecting binding of the binding agent to the protein, thereby codetecting the miRNA and protein in the biological sample.
  • detection is meant detection in the sense of presence versus absence of one or more miRNAs and/or proteins; the registration of the level or degree of expression of one or more miRNAs and/or proteins; and/or the localization of one or more miRNAs and/or proteins at the tissue, cellular or subcellular level.
  • a biological sample as defined herein is a small part of an individual, representative of the whole and may be constituted by a biopsy or a body fluid sample.
  • Biopsies are small pieces of tissue and may be fresh, frozen or fixed, such as formalin- fixed and paraffin embedded (FFPE) .
  • Body fluid samples may be blood, plasma, serum, urine, sputum, cerebrospinal fluid, milk, or ductal fluid samples and may likewise be fresh, frozen or fixed. Samples may be removed surgically, by extraction, i.e., by hypodermic or other types of needles, by microdissection or laser capture .
  • the sample may be any sample as described herein and in certain embodiments is a FFPE sample.
  • in situ method refers to the detection of miRNA and protein in a sample wherein the structure of the sample has been preserved. This may thus be a biopsy wherein the structure of the tissue is preserved.
  • in situ methods are generally histological, i.e., microscopic in nature and include but are not limited to methods such as in situ hybridization techniques and immunohistochemical methods.
  • ISH In situ hybridization
  • ISH is a type of hybridization that uses a complementary nucleic acid to localize one or more specific nucleic acid sequences in a portion or section of tissue ⁇ in situ) , or, if the tissue is small enough, in the entire tissue (whole mount ISH) .
  • RNA ISH is used to assay expression and gene expression patterns in a tissue/across cells, such as the expression of miRNAs/nucleic acid molecules as described herein .
  • the method of this invention is of particular use in the detection of miRNA and proteins in the detection, classification, diagnosis or prognosis of hyperproliferative diseases especially cancers and specifically breast, colorectal, gastrointestinal, lung, pancreas, prostate and/or thyroid cancers.
  • the biological sample is a biopsy sample or a body fluid containing tumor and/or tumor-associated tissue or cells.
  • it may be desirable to obtain more than one sample, such as two samples, such as three samples, four samples or more from individuals, and preferably the same individual .
  • the at least two samples may be taken from normal tissue and hyperproliferative tissue, respectively.
  • a single sample may be compared against a "standardized" sample, such a sample containing material or data from several samples, preferably also from several individuals.
  • a standardized sample may include either normal or hyperproliferative sample material or data.
  • hyperproliferative , and neoplastic refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.
  • Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as nonpathologic , i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of nonpathologic hyperproliferative cells include proliferation of cells associated with wound repair.
  • cancer or "neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal cell carcinoma, prostate cancer, pancreatic and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus .
  • a miRNA or microRNA refers to a 19-25 nt non-coding RNA derived from an endogenous gene that acts as a post-transcriptional regulator of gene expression.
  • MiRNAs are processed from longer (ca 70-80 nt) hairpin-like precursors termed pre- miRNAs by the RNAse III enzyme Dicer.
  • MiRNAs assemble in ribonucleoprotein complexes termed miRNPs and recognize their target sites by antisense complementarity thereby mediating down-regulation of their target genes. Near- perfect or perfect complementarity between the miRNA and its target site results in target mRNA cleavage, whereas limited complementarity between the miRNA and the target site results in translational inhibition of the target gene.
  • the presence, absence, level or localization of any miRNA can be determined.
  • the presence, absence, level or localization of a miRNA of the invention is associated with a hyperproliferative disease, especially cancer, and specifically breast, colorectal, gastrointestinal, lung, pancreas, prostate and/or thyroid cancer.
  • miRNAs include, but are not limited to, those miRNAs exemplified herein (see Table 1) , as well as miRNA including miR 196b (HoxA9) , 10b, pl4, 328, 30A-3P, 125b5, 30E-3P, 680, 134, 604, 128b, 128a, 331, 520F, 299-3P, 520H, 510, 365, 520G, 9, 324-3P, 351, 125A, 146a, 764-5P, 302D, 520D, 652, 520C, 350, 585, 621, 542-5P, 560, 126, and 341.
  • the instant method can be adapted to the detection of virtually any non-coding RNA including small nuclear (snRNAs) , ribosomal RNAs (rRNAs) , transfer RNAs (tRNAs) ultraconserved genetic elements, Piwi- interacting RNAs (piRNA) , small interfering RNAs (siRNA) , and mirtrons.
  • snRNAs small nuclear
  • rRNAs ribosomal RNAs
  • tRNAs transfer RNAs
  • piRNA Piwi- interacting RNAs
  • siRNA small interfering RNAs
  • mirtrons mirtrons.
  • the present invention further includes the detection of other non-coding RNAs such as, but not limited to, snRNA (e.g., U6) , or rRNA (e.g., 18S) in addition to the detection of a miRNA and protein marker.
  • non-coding RNAs are of use in facilitating the quantification of changes in miRNA expression, assessing quality of the biological sample, and serving as an internal control during sample processing and analysis.
  • codetection of other non-coding RNAs and miRNAs may serve to investigate regulatory interactions among these species, or provide for contextualization of the changes of miRNA expression or the other non-coding RNA.
  • a ratio of expression changes of these non-coding RNA species would be more informative than individual detection in independent samples .
  • the method of the invention employs a probe.
  • probe refers to a defined oligonucleotide or a nucleic acid molecule used to detect a target miRNA nucleic acid molecule by hybridization, in particular in situ hybridization.
  • a probe bears a complementary sequence to the target miRNA.
  • Probes of the invention can be single-stranded DNA, double- stranded DNA, RNA or a combination of DNA and RNA.
  • the probe of the invention is synthesized or produced with conventional oligonucleotides. In other embodiments, the probe is modified to include chemical modifications .
  • chemical modifications of a probe can include, singly or in any combination, 2 1 -position sugar modifications, 5-position pyrimidine modifications (e.g., 5- (N-benzylcarboxyamide) -2 1 -deoxyuridine , 5- (N- isobutylcarboxyamide) -2 1 -deoxyuridine, 5- (N- tryptaminocarboxyamide) -2 ' -deoxyuridine , 5- (N- [1- (3- trimethylammonium) propyl] carboxyamide) -2 1 -deoxyuridine chloride, 5- (N-napthylmethylcarboxyamide) -2 1 -deoxyuridine, 5- (Imidazolylethyl) -2 ' -deoxyuridine, and 5- (N- [1- (2 , 3- dihydroxypropyl ) ] carboxyamide) -2 1 -deoxyuridine) , 8-position purine modifications, modifications at exocyclic amines, substitution
  • 5-position pyrimidine modifications refer to pyrimidines with a modification at the C-5 position.
  • Examples of a C-5 modified pyrimidine include those described in US Patent Nos . 5,719,273 and 5,945,527.
  • Examples of a C-5 modification include substitution of deoxyuridine at the C-5 position with a substituent selected from benzylcarboxyamide (alternatively benzylaminocarbonyl ) (Bn) , naphthylmethylcarboxyamide
  • representative C-5 modified pyrimidines include 5- (N- benzylcarboxyamide) -2 1 -deoxyuridine (BndU) , 5- (N- isobutylcarboxyamide) -2 ' -deoxyuridine (iBudU) , 5- (N- tryptaminocarboxyamide) -2 ' -deoxyuridine (TrpdU) and 5- (N- napthylmethylcarboxyamide) -2 ' -deoxyuridine (NapdU) .
  • Modified probes of the invention include substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates , phosphotriesters , phosphoamidates , carbamates, etc.) and those with charged linkages (e.g., phosphorothioates , phosphorodithioates , etc.) , those with intercalators (e.g., acridine, psoralen, etc.) , those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.) , those containing alkylators, and those with modified linkages ⁇ e.g., alpha anomeric nucleic acids, etc.) .
  • internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates , phosphotriesters
  • a modified probe of the invention contains at least one nucleoside analog, e.g., a locked nucleic acid (LNA) .
  • LNA locked nucleic acid
  • the synthesis and preparation of LNA monomers adenine, cytosine, guanine, 5 -methyl -cytosine, thymine and uracil, along with their oligomerizat ion, and nucleic acid recognition properties have been described (Koshkin, et al . (1998) Tetrahedron 54:3607-3630) . LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
  • modifications include 3' and 5' modifications, such as capping.
  • any of the hydroxyl groups ordinarily present in a sugar may be replaced by a phosphonate group or a phosphate group; protected by any suitable protecting groups; or activated to prepare additional linkages to additional nucleotides or to a solid support .
  • the 5 1 and 3 ' terminal OH groups can be phosphorylated or substituted with amines, organic capping group moieties of from about 1 to about 20 carbon atoms, or organic capping group moieties of from about 1 to about 20 polyethylene glycol (PEG) polymers or other hydrophilic or hydrophobic biological or synthetic polymers.
  • PEG polyethylene glycol
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including 2 1 -O-methyl - , 2'-0-allyl, 2'-fluoro- or 2 1 -azido-ribose , carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses , acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • analogous forms of ribose or deoxyribose sugars that are generally known in the art, including 2 1 -O-methyl - , 2'-0-allyl, 2'-fluoro- or 2 1 -azido-ribose , carbocyclic sugar analogs, a-anomeric sugars, epi
  • one or more phosphodiester linkages may be replaced by alternative linking groups.
  • These alternative linking groups include embodiments wherein phosphate is replaced by P (O) S (“thioate”) , P(S)S ("dithioate”) , (0)NR 2 ("amidate”) , P(0)R, P(0)OR', CO or CH 2 ( "formacetal” ) , in which each R or R' is independently H or substituted or unsubstituted alkyl (C1-C20) optionally containing an ether (-0-) linkage, aryl , alkenyl, cycloalkyl, cycloalkenyl or araldyl . Not all linkages in a probe need be identical. Substitution of analogous forms of sugars, purines, and pyrimidines can be advantageous in designing a final product, as can alternative backbone structures like a polyamide backbone, for example.
  • a modification to the nucleotide structure of a probe may be imparted before or after assembly of the probe.
  • a sequence of nucleotides may be interrupted by non-nucleotide components.
  • a probe may be further modified after polymerization, such as by conjugation with a labeling component.
  • the probe has a nucleotide sequence selected from the group of SEQ ID NOs : 1 to 56, or a fragment thereof that can hybridize under stringent condition, and/or has an identity of at least 80% to any of these sequences.
  • the probe is selected from the group of SEQ ID NOs: 2, 5, 8, 9, 10, 13, 16, and 18, or a fragment thereof that can hybridize under stringent conditions, and/or has an identity of at least 80% to any of these sequences.
  • a probe is detected with a label or tag or otherwise modified to facilitate detection.
  • a label or a tag is an entity making it possible to identify a compound to which it is associated. It is within the scope of the present invention to employ probes that are labeled or tagged by any means known in the art such as, but not limited to, radioactive labeling, affinity labeling (e.g., with a hapten and its associated antibody) , fluorescent labeling and enzymatic labeling.
  • the probe may be immobilized to facilitate detection.
  • a hapten is incorporated into the probe of the invention.
  • Haptens commonly employed in labeling applications include fluorescein (e.g., 5- or 6 -carboxy- fluorescein, FAM) , biotin, digoxigenin (DIG), 5 -bromo-2 -deoxyuridine (BrdU) and dinitrophenol .
  • FAM fluorescein
  • DIG digoxigenin
  • BrdU 5 -bromo-2 -deoxyuridine
  • Probe synthesis and hapten incorporation are routinely practiced in the art and any suitable method can be employed. See, e.g., Luehrsen, et al . (2000) J. Histochem. Cytochem. 48:133-145.
  • the probe contains a hapten that is detectable using an immunoassay.
  • certain embodiments of this invention include the use of an anti -hapten antibody.
  • binding of the probe to the miRNA can be detected by contacting the hapten with an anti -hapten antibody, contacting the anti -hapten antibody with a secondary antibody reagent, and detecting the secondary antibody reagent by routine methods as described herein.
  • a secondary antibody reagent is composed of an antibody covalently linked to a protein that provides for a detectable signal .
  • Suitable detectable proteins include, but are not limited to, fluorescent proteins, chromogenic proteins, and enzymes that catalyze the production of a product that is luminescent, fluorescent, or colored (e.g., ⁇ - galactosidase , luciferase, horse radish peroxidase, alkaline phosphatase, etc.) .
  • Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP; Chalfie, et al .
  • BCIP nitro-blue tetrazolium chloride
  • NBT nitro-blue tetrazolium chloride
  • certain embodiments of the present invention embrace the use of species and/or isotype-specific secondary antibodies.
  • the detection of one marker can be achieved with an anti -mouse secondary antibody, whereas the detection of a second marker can be achieved with an anti-rabbit secondary antibody.
  • the detection of one marker can be achieved with an anti-IgGl- specific mouse secondary antibody, whereas the detection of a second marker can be achieved with an anti-IgG2a specific secondary antibody.
  • Fluorescent compounds containing fluorophores also known as fluorochromes
  • Fluorescein and rhodamine usually in the form of isothiocyanates that can be readily coupled to antigens and antibodies, are most commonly used in the art (Stites, et al . (1994) Basic and Clinical Immunology) .
  • Fluorescein absorbs light of 490 nm to 495 nm in wavelength and emits light at 520 nm in wavelength.
  • Tetramethylrhodamine absorbs light of 550 nm in wavelength and emits light at 580 nm in wavelength.
  • Illustrative fluorescence-based substrates are described herein and further include such examples as ELF- 97 alkaline phosphatase substrate (Molecular Probes, Inc., Eugene, OR); PBXL-1 and PBXL-3 (phycobil isomes conjugated to streptavidin) ; and CY substrates.
  • ELF-97 is a nonfluorescent chemical that is digested by alkaline phosphatase to form a fluorescent molecule. Because of turnover of the alkaline phosphatase, use of the ELF-97 substrate results in signal amplification.
  • Illustrative luminescence-based detection reagents include CSPD and CDP star alkaline phosphatase substrates (Roche Molecular Biochemicals, Indianapolis, IN) ; and SUPERSIGNAL horseradish peroxidase substrate (Pierce Chemical Co., Rockford, IL) .
  • the present method also features the codetection of a protein in the biological sample.
  • Proteins that can be detected in accordance with the present invention include, but are not limited to, those proteins exemplified herein (see Table 3) , as well as other proteins including COX-2, pl6, CD73, CD138, notch receptor-3, CD90, BMI-1, IGF2 , YKL-40, EGF-R, c-jun, PCNA, JNK, cyclin Bl, c-kit, STAT3 , cyclin Dl, PI3K, MAPK, MAPKK, DDR2, TRF2, activin, EGFR, HER- 2 , HER- 3 , HER-4, MEKl/2 and/or phosphorylated or post -translationally modified versions of said proteins, e.g., phosphorylated EGFR (pEGFR) ,
  • the protein being detected is a cell type-specific protein marker or functional marker, i.e., a protein that distinguishes one cell type from another.
  • Ki- 67 is a known marker for proliferative cells
  • CK19 is a known epithelial marker for cancer cells (of epithelial origin; carcinomas)
  • CK7 and CK20 are known differential markers of lung and colorectal cancer cells (of epithelial origin)
  • CD31 is an endothelial marker
  • CK5/6 has been shown to be a reliable marker for mesothelioma and squamous cell carcinoma of the lung as well as a marker for the aggressive basal (ER- PR-HER2 - ; triple negative) subtype of breast carcinoma.
  • cell-type specific protein markers are well-known in the art and can be detected in accordance with the present method.
  • a protein can be readily detected by a binding agent such as ligand or antibody that specifically binds the protein of interest with little to no detectable binding to any other protein in a sample.
  • the binding agent is an antibody specific for the protein of interest.
  • Antibodies to the proteins disclosed herein are well-known in the art and available from a number of commercial sources .
  • binding agent is a ligand of the protein of interest
  • said ligand can be labeled or tagged as described herein and detected via an antibody.
  • the binding agent is an antibody (i.e., a primary antibody)
  • a secondary antibody reagent ⁇ e.g., an enzyme-conjugated secondary antibody
  • binding of the primary antibody to the protein of interest is detected by contacting the primary antibody with an enzyme-conjugated secondary antibody, and detecting enzyme activity by routine methods as described herein.
  • Exemplary enzyme- conjugated antibodies of use in the claimed method include, but are not limited to, horseradish peroxidase-conjugated antibodies and alkaline phosphatase-conjugated antibodies.
  • certain embodiments of this invention further include the use of epitope retrieval methods to facilitate detection of the protein of interest.
  • epitope retrieval methods include, but are not limited to, heat-induced epitope retrieval with or the use of commercially available reagents, e.g., Reveal (Biocare Medical) or other pH buffered reagents.
  • the instant method finds application in the detection of multiple miRNAs and proteins by sequential rounds of detecting individual miRNAs and proteins with different fluorescent substrates (see, e.g., Figure 5).
  • the present method includes inactivation of the label of the probe and, in some embodiments, inactivation of the secondary antibody reagent.
  • the label of the probe is covalently attached to the probe, e.g., via a protease cleavable linker, said linker can be cleaved to remove the label from the probe thereby inactivating the label.
  • the label is a hapten and its associated anti-hapten antibody and enzyme -conjugated secondary antibody specific for the anti-hapten antibody
  • the label can be rendered inactive by destroying the activity of the enzyme.
  • sodium azide or hydrogen peroxide is commonly used to block horseradish peroxidase activity and can be used in accordance with the instant method.
  • a secondary antibody reagent such as horseradish peroxidase- conjugated secondary antibody
  • said secondary antibody can likewise be inactivated by sodium azide or hydrogen peroxide.
  • the method of the invention when detecting multiple miRNAs and proteins in a biological sample, is modified to include the steps of (a) contacting a biological sample with a probe that binds to a miRNA, (b) detecting binding of the probe to the miRNA with a label, (c) inactivating the label, (d) contacting the biological sample with an antibody that specifically binds to a protein, (e) detecting binding of the antibody to the protein with a secondary antibody reagent, (f) inactivating the secondary antibody reagent, and (g) repeating steps (a) to (f) until each of the miRNA and protein of interest have been detected.
  • the miRNA and protein are both detected via the use of enzyme-conjugated secondary antibodies, which can be inactivated between each detection step, it will be readily appreciated by the skilled artisan that there is no particular order in which the miRNA and protein are detected; the miRNA can be detected first or the protein can be detected first.
  • particular embodiments embrace the use of enzyme substrates with different fluorophores .
  • the present invention also features a kit, container, pack, or dispenser containing probes and binding agents of the invention together with instructions for use.
  • the kit may be for the detection of a miRNA and protein, or for the classification, diagnosis and/or prognosis of a disease related to the miRNA and/or protein such as a hyperproliferative disease, e.g. a cancer.
  • the kit can include the probes and binding agents of the invention in a form suitable for the detection of said miRNA and protein.
  • the kit may include any reagent for the detection of said probes or binding agents, e.g., enzyme-conjugated secondary antibodies and substrates.
  • the kit includes a probe selected from the group of SEQ ID NO : 1 to 56.
  • the probe is selected from the group of SEQ ID NOs: 2, 5, 8, 9, 10, 13, 16, and 18.
  • the kit also includes instructions for hybridizing the probe to the miRNA and the binding agent to the protein. Instructions can include a detailed or step-by-step protocol, including hybridization conditions, to perform the method of the invention.
  • the instructions can be provided as a text file or printout, or when the kit is compatible with an automated staining system, the instructions can be provided as computer file containing the protocol program.
  • FITC fluorescein
  • tyramide signal amplification (TSA) reaction in which horseradish peroxidase (HRP) was conjugated to an anti-FITC antibody (which binds to the FITC-labeled miRNA probe) activated the tyramide moiety of a fluorescent substrate resulting in a covalent attachment to proteins in the vicinity of the miRNA probe.
  • HRP horseradish peroxidase
  • anti-FITC antibody which binds to the FITC-labeled miRNA probe
  • the instant method has many clinical applications for disease management. Specifically, using the combined ISH/IHC assay, the expression of a panel of cancer-associated miRNAs in archived FFPE specimens of breast, colorectal, lung, pancreatic and prostate carcinomas were analyzed. The results uncovered a complex and distinct contribution of different cell types to altered expression of individual miRNAs in tumor tissues.
  • the observations herein indicate that altered miRNA expression may not be confined to cancer cells to be etiologically relevant (e.g., miR-21 in tumor associated fibroblasts) , whereas other miRNA expression changes may simply reflect tissue heterogeneity (e.g., endocrine cell- expressed miR-375 in pancreatic ductal adenocarcinoma) .
  • tissue heterogeneity e.g., endocrine cell- expressed miR-375 in pancreatic ductal adenocarcinoma
  • Co- registration with cell type-specific protein markers indicated a previously unappreciated contribution of tumor microenvironment elements to altered miRNA expression.
  • the source cell (s) of altered miRNA should be carefully considered when designing and interpreting miRNA- based diagnostic assays.
  • results herein also have implications for miRNA-based therapeutic interventions aimed at restoring basal miRNA activity; since the source cell (s) of miRNA deregulation could be the cancer cells, reactive stroma, infiltrating immune cells and/or other involved cell types, different targeted delivery strategies may be required for different miRNAs (Sempere & Kauppinen (2009) supra) .
  • These findings underscore the necessity of spatial characterization of miRNA expression to determine whether the cancer cells, supportive and/or reactive microenvironment elements are the principal source of miRNA deregulation in solid tumors. This information is essential to understand the role of miRNAs in cancer initiation and progression as well as to interpret the impact of miRNA changes in a diagnostic test. Accordingly, the instant method finds application in the detection, classification, prevention, diagnosis, prognosis as well as treatment of cancer .
  • Tissue specimens were obtained through the research pathology services from the Tissue Bank at Dartmouth- Hitchcock Medical Center, Riverside, NH. These surgical specimens were processed in the surgical pathology laboratory using an institutional standardized protocol. Briefly, surgical specimens were sectioned in 2 mm slices and fixed in 10% formalin for up to 24 hours and then were paraffin-embedded in fully-automated Shandon Pathcentre instrument using a standard overnight procedure (2 x 10% formalin for 80 minutes, ethanol series 75% to 100 in 6 sequential 45 -minute steps, 2 x xylenes for 45 minutes and 4 x paraplast for 45 minutes) .
  • LNA- modified DNA probes were designed to have a predicted melting temperature (T m ) between about 75°C to 78°C (see Table 1) following the general strategy as previously described (Valoczi, et al . (2004) Nucleic Acids Res. 32 (22) : el75) . Briefly, LNA-modified nucleotides were intercalated at every third nucleotide among DNA nucleotides. When this strategy rendered a probe with a T m lower than 70°C, more LNA-modified nucleotides were introduced, which were intercalated at every second or third position. C or G for LNA modifications were preferentially selected.
  • the T m of the probe against complementary miRNA sequence was calculated using LNA design SciTools (Integrated DNA Technologies Inc., Coralville, IA) , with default parameters, except for [Na+] which was set to 100 mM.
  • these are the codes for the hapten addition onto a terminal 5' and/or 3' extra T nucleotides (these Ts are not part of the complementary miRNA sequence) : /5biosg/t, /55Br-dU/tt, /5DigN/t, /56-FAM/t, /5Biosg/t [ ... ] t/3Bio/ , /55Br- dU/tt [ ...
  • let-7a 78.3 AA+CTA+TA+CAA+CC+TA+CTA+CCT+CA 1 miR-21 73.8 T+CAA+CAT+CA+GT+CTG+ATA+AG-iCTA 2 miR-24 80 CT+GTT+CCT+GCT+GAA+CTG+AGC+CA 3 miR-34a 80 A+CAA+CCA+GCT+AAG+ACA+CTG+CCA 4 miR-34c-5p 79 GCA+ATC+AGC+TAA+CTA+CA+CTG+CCT 5 miR-125b 77.2 TC+ACA+AGT+TAG+GGT+CTC+AGG+GA 6 miR-126 73.4 CG+CAT+TAT+TAC+TCA+CGG+TAC+GA 7 miR-141 75 C+CAT+CTT+TAC+CA+GA+CA+GTG+TTA 8 miR-143 73.4 GA+GCT+ACA+GTG+CTT+CAT+CA 9 miR-143
  • tissue was mounted on positively-charged barrier frame slides, de- waxed in xylenes, and re-hydrated through an ethanol dilution series (100% to 25%) . Tissue sections were digested with 5 yg/mL of proteinase K for 20 minutes at 37°C to facilitate probe penetration and exposure of miRNA species. To minimize non-specific binding based on charge interactions, tissue was subject to a brief acetylation reaction (66 m HC1 , 0.66% acetic anhydride (v/v) and 1.5% triethanolamine (v/v) in RNase-free water) .
  • acetylation reaction 66 m HC1 , 0.66% acetic anhydride (v/v) and 1.5% triethanolamine (v/v) in RNase-free water
  • tissue sections were pre-hybridized at the hybridization temperature (see Table 2) for 30 minutes in pre- hybridization solution, which was composed of 50% deionized formamide, 5X Sodium chloride-Sodium citrate buffer (SSC) , IX Denhardt's solution, 500 g/ml yeast tRNA, 0.01% TWEE .
  • Pre-hybridization solution was replaced with 200 ih of hybridization solution containing 10 pmol of the hapten- labeled LNA probe and tissue were incubated for 90 minutes at T hyb and washed three times for 10 min in SSC buffer at the established stringency of SSC (see Table 2 for details) .
  • miR-21 45 0.5X TAFs, cancer cells miR-24 37 0.5X Various
  • miR-145 50 0.
  • IX Smooth Muscle Cells miR-155 50 0.5X
  • Leukocytes miR-196 37 0.5X Cancer cells miR-200b
  • Various miR-205 50 0.
  • IX Myo/basal epithelia miR-214 50 0. IX Various
  • AMCA 1 300-1 : 500 15 min. (10-30 min. )
  • Rhodamine 1 500 15 min. (10-30 min. ) TABLE 7
  • ALEXA fluor 647-NHS ester was obtained from Invitrogen; AMCA-NHS ester, DYLIGHT405 -NHS ester, DYLIGHT594 -NHS ester, DYLIGHT649-NHS ester, DYLIGHT680 -NHS ester, Fluorescein-NHS ester, and Rhodamine-NHS ester were obtained from Pierce; and Tyramine hydrochloride, dimethyl formamide (DMF) , and triethylamine (TEA) were obtained from Sigma. For acetylation reactions, triethanolamine was used.
  • Tissue availability were limited such as tissue cores of a tissue microarray, core needle biopsies, and cell preparations obtained by fine needle aspiration; and/or ii) Multivariate analysis of miRNAs with opposite expression changes between normal and cancer cells or within different cell types within the tumor lesion yielded a more powerful clinical indicator than either miRNA alone.
  • probes were utilized that were 5' and 3' terminally tagged (hapten2X) with biotin (Bio)-, digoxigenin (DIG)-, 5 -bromo-2 -deoxyuridine (BrdU) - or 5- (and-6) -Carboxyfluorescein (FAM) .
  • Bio biotin
  • DIG digoxigenin
  • BadU 5 -bromo-2 -deoxyuridine
  • FAM Carboxyfluorescein
  • CK19 Cytokeratin 19
  • CK5/6 and CK14 are expressed in normal myoepithelial cells and specifically identify the aggressive basal breast cancer subtype (Moriya, et al . (2006) Med. Mol . Morphol .
  • CK7/CK20 signal can be used to discern the organ of origin of certain cancers, for example most primary and metastatic colorectal carcinoma cells are CK7-/CK20+ and lung carcinoma cells are CK7+/CK20- cells (Tot (2002) Bur. J. Cancer 38(6) :758-63); iv) Glucagon and insulin are specifically expressed in a and ⁇ endocrine pancreatic cells, respectively, and as such are useful markers for establishing the type of endocrine pancreatic tumors (Tomita (2002) Pathol. Int. 52 (7) : 425-32) .
  • miRNAs have been shown to mediate the repression of and/or to be regulated by important oncogenic and tumor suppressor genes (Ventura & Jacks (2009) supra; Sempere & Kauppinen (2009) supra) .
  • codetection of miRNA and protein markers have important clinical applications.
  • Detection of protein markers was generally compatible with proteinase K digestion and other chemical treatments used in the preceding ISH steps.
  • Some useful epithelial-specific markers such as CK19 and other cytokeratins (e.g., CK7 , CK8/18 and CK20) were detected without further tissue processing, while other protein markers required heat -induced epitope retrieval (HIER) to be efficiently detected.
  • HIER heat -induced epitope retrieval
  • the mildest proteinase K treatment (limit incubation time) and HIER treatment (limit the temperature of HIER and/or incubation time) should be used for optimal codetection conditions of miRNAs and proteins.
  • HIER treatment limit the temperature of HIER and/or incubation time
  • Table 1 also lists a general summary of the expression pattern of the 18 miRNAs that were analyzed in this study.
  • ISH/IHC assay for detection of miR-205 and basal/squamous epithelial cell marker would increase the interpretative power of miR-205 expression changes and could be further implemented as a diagnostic and/or prognostic tool in solid tumors.
  • ISH results herein indicate that miR-126 expression changes in tumor tissue could mainly reflect differences in distribution of blood vessels compared to normal tissue, and perhaps, differences in the integrity and/or structure of the neovasculature .
  • expression profiling analyses detected miR-375 at lower levels in pancreatic tumor tissues compared to normal pancreas (Mardin & Mees (2009) Ann. Surg. Oncol. 16 (11) : 3183-9) .
  • ISH results herein indicate that miR-375 is expressed in cell types that do not contribute to pancreatic ductal adenocarcinoma formation .
  • miR-141 was reported as a potential serum biomarker for prostate cancer detection (Mitchell, et al .
  • codetection of miR-21, p53 and E-cadherin are of use in the diagnosis of colon cancer.
  • MiR-34 family members are transcriptionally activated by the p53 tumor suppressor gene in response to DNA damage and mitogenic signals (He, et al . (2007) Nat. Rev. Cancer 7 (11) : 819-22) . Consistent with a tumor suppressive role, miR-34s were detected at lower levels in breast, lung and other solid tumors by expression profiling analyses in whole tissue biopsies (Barbarotto, et al . (2008) supra) .
  • Example 8 miRNAs and the Immune Response
  • ISH is not as sensitive as other detection methods, particularly real time RT-PCR, undetectable or low levels of expression of miR-155 by ISH may still be functionally important within cancer cells.
  • Recent studies reported detection of miR-155 by ISH within immune cells and cancer cells in colorectal adenocarcinoma lesions and predominantly within cancer cells in pancreatic adenocarcinoma lesions (Valeri, et al . (2010) supra; Ryu, et al. (2010) Pancreatology 10 (1) : 66-73) .
  • miR-21 Like miR-155, high levels of miR-21 expression have been frequently detected in hematologic and solid tumors and miR-21 is considered to be an important oncogenic miRNA based on functional studies in cancer cell lines and animals models (Sempere & Kauppinen (2009) supra; Krichevsky & Gabriely (2009) J " . Cell. Mol . Med. 13(1) :39- 53) . However, it was found that in solid tumors the source cells of altered expression for miR-21 and miR-155 were strikingly different. Higher levels of miR-21 expression were detected within cancer cells and tumor-associated fibroblasts (TAFs) compared to matched normal tissues.
  • TAFs tumor-associated fibroblasts
  • miR-21 was predominantly expressed at higher levels within cancer cells in lung, pancreas and prostate cancer; whereas in breast and colorectal cancer, higher levels of miR-21 expression were more apparent within tumor associated fibroblasts (TAF) as supported by co-staining with the mesenchymal markers vimentin and smooth muscle actin (Figure 4) .
  • TAF tumor associated fibroblasts
  • Figure 4 These observed patterns of miR-21 accumulation were consistent with previous studies in breast and lung cancer and an independent study in colorectal adenocarcinoma (Sempere, et al . (2007) supra; Yamamichi, et al . (2009) supra; Liu, et al . (2010) supra), but were in disagreement with reported nuclear-enriched staining of miR-21 within cancer cells in pancreatic adenocarcinoma lesions (Dillhoff, et al . (2008) supra) .
  • miR-146a expression has been correlated with cervical cancer.
  • Pancreatic ductal adenocarcinoma is the fourth- leading cause of cancer related mortality in the US with a median survival rate of 6 months.
  • PDAC's lethal nature stems from its unique biology: aggressive local invasion, marked desmoplasia, resistance to apoptosis and chemotherapy, and metastatic potential .
  • Non-coding microRNAs are involved in initiation and dissemination of many types of cancer, including PDAC.
  • MiR-lOb induced by the transcription factor Twist, has been found to target the expression of HOXD10, promoting epithelial to mesenchymal transformation (EMT) .
  • miR-lOb expression was analyzed in PDAC by comparing the spatial expression of miR-lOb in PDAC with the normal pancreas, intraductal papillary mucinous neoplasms (IPMNs) , benign cysts, and neuroendocrine tumors.
  • IPMNs intraductal papillary mucinous neoplasms
  • miR- 10b levels are highest in PDAC, intermediate in IPMN, and lowest in benign cysts. These findings indicate that miR- 10b could serve as a novel biomarker for the metastatic potential of PDAC and for the malignant transformation of IPMN.
  • Expression profiles of miRNAs can provide prognostic information in lung cancer.
  • Specific miRNA expression patterns can assess survival outcomes for lung cancer patients.
  • a 4-miRNA (miR-486, miR-30d, miR-1, and miR-499) serum signature in NSCLC can predict overall survival (Hu, et al . (2010) J " . Clin. Oncol. 28:1721-6).
  • Increased miR-155 and reduced let-7a-2 expression defines an unfavorable survival in pulmonary adenocarcinomas (Yanaihara, et al . (2006) Cancer Cell 9:189-98) .
  • lung-cancer-associated miRNAs which are biomarkers of treatment response or pharmacological targets. Given these data, searches were undertaken to find those miRNAs that would exert tumor- suppressive or oncogenic (oncomir) effects in the lung.
  • Candidate tumor-suppressive miRNAs are those that exhibit a reduced expression in malignant versus adjacent normal lung tissues.
  • Potential oncomirs are defined by overexpression in the malignant versus adjacent normal lung tissues. Consequences of this overexpression include changes in the expression of critical target genes that could confer tumor-suppressive or oncogenic effects of specific miRNAs. Changes in these direct target genes can be used to improve the diagnosis or classification of lung cancers. Some of these species are also therapeutic or chemopreventive targets in the lung, as in the case of miR-31 (Liu, et al . (2010) J " . Clin. Invest. 120:1298-309).
  • Oncomirs in Lung Cancer have a higher basal expression in malignant as compared with adjacent normal lung tissues.
  • few candidate oncomirs have had mechanistic validation or identification of the target genes that would exert their oncogenic effects.
  • miR-21 plays a key functional role in several cancers (Corsten, et al . (2007) Cancer Res. 67:8994-9000; Asangani , et al . (2008) Oncogene 27:2128-36; Ribas , et al . (2009) Cancer Res. 69:7165-9; Wang, et al . (2009) Cancer Res. 69:8157-65) including lung cancer (Seike, et al . (2009) Proc . Natl. Acad. Sci. USA 106:12085-90).
  • miR-21 targets multiple negative regulators of the Ras/methyl ethyl ketone/extracellular receptor kinase pathway to promote proliferation by regulating the expression of Spryl, Spry2 , Btg2 , and Pdcd4 (Zhu, et al . (2008) supra; Hatley, et al . (2010) Cancer Cell 18:282-93; Lu, et al . (2008) Oncogene 27:4373-9) .
  • miR-21 inhibits apoptosis by reducing the expression of proapoptotic gene products that include Apafl, Faslg, Pdcd4, and RhoB (Zhu, et al . (2008) supra; Hatley, et al . (2010) supra) . Reducing Pdcd4 expression can increase invasion and metastasis (Lu, et al . (2008) supra) .
  • miR-31 acts as an oncomir in murine and human lung cancers (Liu, et al . (2010) J “ . Clin. Invest. 120:1298-309) .
  • the overexpression of miR-31 and other miRNAs in transgenic cyclin E-driven murine lung cancers implied that a similar miRNA expression profile would occur in human lung cancers (Ma, et al . (2007) Proc . Natl. Acad. USA 104:4089-94) . This was the case when human lung cancers (vs. adjacent normal lung tissues) had a similar expression pattern for several miRNAs that were highlighted in previously described transgenic lung cancers (Liu, et al .
  • regulating miR-31 expression also affects breast cancer metastasis by regulating the expression of other target genes (Valastyan, et al . (2010) Cancer Res. 70:5147-54; Valastyan, et al . (2009) Cell 137:1032-46) .
  • these findings underscore that miRNAs exert their functions in cell-, tissue-, and disease-specific contexts.
  • Pharmacologic knock-down of a critical oncogenic miRNA, such as miR-31 in lung cancer might exert antineoplastic effects (Liu, et al . (2010) supra) .
  • miR-146b expression patterns in squamous cell lung cancer predicted a poor clinical outcome
  • miR-34a expression was identified as a biomarker for clinical relapse in surgically resected NSCLC (Raponi, et al . (2009) Cancer Res. 69:5776-83; Gallardo, et al . (2009) Carcinogenesis 30:1903-9)
  • miR-34a was inactivated by CpG methylation, which caused transcriptional silencing in lung cancers (Ladygin, et al . (2008) Cell Cycle 7:2591- 600) . The precise mechanisms through which these miRNAs exert their tumor- suppressive effects remain to be determined .
  • the miR-34 family is reported as a p53-induced tumor- suppressive miRNA family in diverse types of cancers (He, et al. (2005) Nature 435:828-33; Change, et al . (2008) Nat. Genet. 40:43-50; Corney, et al . (2007) Cancer Res. 67:8433-8; Welch, et al . (2007) Oncogene 26:5017-22; Tazawa, et al . (2007) Proc . Natl. Acad. Sci . USA 104:15472- 7; Bommer, et al . (2007) Curr. Biol. 17:1298-307) .
  • p53 directly induces miR-34 family transcription. Ectopic miR-34 expression can augment apoptosis, cell -cycle arrest, or senescence.
  • the promoter regions of the miR-34 family often are inactivated by CpG methylation (Lodygin, et al . (2008) supra) . Repression of the miR-34 family was linked to a resistance to p53- activating agents that can cause apoptotic response to specific chemotherapy treatments (Zenz, et al . (2009) Blood 113:3801-8) .
  • Direct miR-34 target genes include CKD4/6 (Fujita, et al . (2008) Biochem. Biophys . Res. Commun.
  • the let-7 miRNA family is located in genomic regions that are fragile sites or frequently are deleted in specific cancers (Calin, et al . (2004) Proc . Natl. Acad. Sci. USA 101:2999-3004). Let-7 expression is often repressed in certain types of lung cancers (Yanaihara, et al . (2006) supra; Inamura, et al . (2007) supra; Takamizawa, et al . (2004) supra) . Engineered let-7 overexpression can inhibit cancer cell proliferation (Lee & Dutta (2007) Genes Dev. 21:1025-30; Johnson, et al . (2005) Cell 120:635-47) .
  • let-7 target genes include K-ras (Johnson, et al . (2005) supra), HMGA2 (Lee & Dutta (2007) supra; Hebert, et al . (2007) Mol . Cancer 6:5), and c-Myc21 (Koscianska, et al . (2007) BMC Mol. Biol. 8:79), as well as the cell-cycle regulators CDC25A, CDK6 , and cyclin D2 (Johnson, et al . (2007) Cancer Res. 67:7713-22) .
  • let-7 Engineered overexpression of let-7 was achieved in the K-ras-driven murine lung cancer model via adenoviral delivery, which reduced lung cancer formation in vivo (Kumar, et al . (2007) Nat. Genet. 39:673-7) . Forced overexpression of tumor- suppressive miRNAs can exert antineoplastic effects in lung cancer (Liu, et al . (2009) supra) .
  • miRNA signatures for each histopathologic subtype of lung cancer can be assessed and compared to any changes relative to the normal lung in addition to the clinical features present at diagnosis such as stage, smoking history, age, and gender.
  • miRNA profiles can prove informative in predicting the response to chemotherapeutic or targeted therapies.
  • miRNA profiles can identify those at high risk for developing lung cancer. This information can guide the use of chemopreventive agents to reduce this clinical risk.
  • miRNA profiles exert biological effects by regulating the expression of many target genes. Bioinformatic analysis can predict potential target genes for each miRNA (Lewis, et al . (2003) Cell 115:787-98; Lewis, et al . (2005) Cell 120:15-20). Functional validation of any highlighted target gene can be confirmed by establishing the direct complex formation of the miRNA of interest with the expected mRNA sequence. In these analyses, miRNA profiles could depend on the cell and tissue contexts as well as on the examined physiological or pathophysiological states. It is notable that a change in a single miRNA can lead to compensatory and time-dependent changes in protein (or mRNA) expression profiles within individual cells or tissues (Selbach, et al .
  • Example 12 MiRNA as Biomarkers for Management of Breast Cancer
  • Change column refers to miRNA expression change between normal and tumor breast tissue using total RNA extracted from whole tissue using different detection tools (e.g., microarray profiling and/or RT-PCR) ; accordingly “Down” refers to decrease detection and “Up” to increase detection of miRNA levels in tumor tissue.
  • detection tools e.g., microarray profiling and/or RT-PCR
  • “Change” column refers to expression changes specifically within epithelial cells. --, to be determined. ⁇ " ⁇ ISH data from Sempere, et al . (2007) supra.
  • ISH in situ hybridization
  • the tissue was pre-hybridized at the hybridization temperature (Thyb: 25°C below calculated T m of the probe) for 30 minutes in hybridization solution, which was composed of 50% deionized formamide, 5X Sodium chloride- Sodium citrate buffer (SSC) , IX Denhardt's solution, 500 ⁇ q/ml yeast tRNA, 0.01% TWEEN. Subsequently, this hybridization solution was replaced with hybridization solution containing 10 pmol of the hapten-labeled LNA probe and incubated for 90 minutes. Tissue slides were washed three times for 10 minutes in SSC buffer at the established stringency of SSC (between IX to 0.2X) at Thyb.
  • SSC Sodium chloride- Sodium citrate buffer
  • slides were loaded onto the i6000 staining machine, which automatically dispensed 400 pL per slide.
  • the machine washed the slides with phosphate-buffered saline and 0.01% TWEEN (PBST) solution.
  • Slides were subsequently treated with 3% H 2 0 2 to inactivate endogenous peroxidase and blocked with bovine serum albumin (BSA) to minimize non-specific binding of the rabbit anti-hapten/HRP antibody with or without further amplification with anti- rabbit/HRP secondary antibody.
  • BSA bovine serum albumin
  • the tyramine-conjugated fluorochrome was applied to the slide and the TSA reaction proceeded for 10-30 minutes.
  • slides were profusely washed with PBST and were ready to be mounted on anti-fading PROLONG gold solution
  • miRNA Expression in Breast Cancer Samples Lower detection of miR-451 levels in whole tumor tissue compared to normal by microarray profiling did not confirm to be etiologically relevant, but rather it indirectly reflected architectural changes of the tumor-associated vasculature; miR-451 was predominantly expressed in mature erythrocytes as determined by ISH and independently confirmed by functional assays of hematopoietic cell differentiation (Zhan, et al . (2007) Exp. Hematol . 35:1015-1025; Rathjen, et al. (2006) FEBS Lett. 580:5185-5188).
  • miR-145 and miR-205 expression was restricted to myoepithelial cells in normal epithelial structures, whereas their expression was reduced or completely eliminated in matching tumor specimens.
  • expression of let-7a, miR-21, miR-141, miR-214 was detected at varying levels predominantly within luminal epithelial cells in normal tissue. Let-7a expression was consistently decreased within cancer cells, while miR-21 expression was frequently increased within cancer cells, but also in tumor-associated fibroblasts.
  • miR-141 and miR-214 exhibited a more complex pattern requiring a larger sample size to determine whether their expression changes are associated with any clinical parameters.
  • the most significant associations that were identified were early manifestation of altered miR-145 expression in carcinoma in situ lesions adjacent to invasive carcinoma and thus presumed pre- invasive ; and high miR-205 expression correlates with favorable clinical outcome in ER-PR-HER2 - cases, although in a limited sample of 20 cases.
  • miR-lOb expression may be restricted to and be etiologically relevant in cells at advancing edge of the tumor and lamented that whole tissue profiling may confound the signal of miRNAs such as miR-lOb expressed in a restricted number of cells (Ma, et al . (2007) Nature 455 :E8-E9) .
  • miR- 34a Proxy Indicator of Tumor Suppressive Pathway.
  • miR- 34a was predominantly expressed in luminal epithelial cells and its expression was markedly reduced in carcinoma in situ (CIS) and invasive carcinoma (lea) lesions. Therefore, miR-34a could be useful as an indicator of progression from non- invasive to preinvasive disease within a different epithelial subpopulation .
  • miR-34a is of particular interest given the well-established transcriptional activation of miR-34 by tumor suppressor gene p53 and its pro-apoptotic and anti -mitogenic role in cancer cells (He, et al . (2007) Nature 435:828-833) .
  • p53 expression detected by IHC and/or p53 sequencing analysis may not necessarily reflect loss of p53 activity status, which is associated with poor prognosis (Troester, et al . (2006) BMC Cancer 6:276) . Since inactivation of p53 is not always associated with loss of p53 expression, miR-34a levels could inform by proxy of p53 activity status. Thus, miR-34 and other miRNAs could serve as powerful indicators of the status of tumor suppressive or oncogenic pathways.
  • miR-210 was detected in epithelial cells and stroma in normal breast tissue, and expression of miR-210 persisted in matched CIS and ICa lesions. miR-210 has been suggested as an independent prognostic indicator and upregulation of miR-210 expression correlates with hypoxia (Camps, et al . 92008) Clin. Cancer Res. 14 : 1340-1348) .
  • miR-125b accumulated at higher levels in myoepithelial cells and surrounding fibroblasts in normal breast tissue.
  • the cancer cells with higher levels of miR-125b could be more susceptible to herceptin treatment since miR-125b and herceptin may act cooperatively to disrupt HER2 signaling and thus miR-125b expression could be useful as predictor of treatment response in HER2+ cases .
  • Endoglin or CD105 is a cell surface glycoprotein and functions as a co-receptor for TGF- ⁇ and it is upregulated in endothelial cells of neovasculature (Duff, et al . (2003) FEBS Lett. 17:984-992) .
  • a high number of CD105-positive blood vessels and high levels of CD105 in serum are associated with poor prognosis and decreased treatment response (Beresford, et al . (2006) Br. J. Cancer 95:1683-88; Vo, et al . (2008) Breast Cancer Res. Treat. 119 (3) : 767-71; Kumar, et al . (1999) Cancer Res. 59:856-861) .
  • miR-126 may be a useful marker to interpret CD105 staining and refine the assessment of vascular normalization. This also points to the fact that miRNA changes could be clinically informative in cell types other than the cancer cells per se .
  • miR-21 and miR-125 negatively regulate expression of PTEN (Meng, et al . (2007) Gastroenterology 133:647-658) and HER2/HER3 (Scott, et al . (2006) supra) in breast cancer cell lines, respectively.
  • PTEN PTEN
  • HER2/HER3 Scott, et al . (2006) supra
  • miR-21/PTEN and miR-125/HER-2/3 interactions are operational in tumors as suggested by these in vitro experiments, and could notably affect the efficacy of herceptin treatment.
  • these miRNAs are informative for HER2+ tumors.
  • miR-125 could cooperate with herceptin to inhibit HER2/3 signaling, whereas miR-21 would oppose herceptin action by inhibiting PTEN, the natural suppressor of PI3K/AKT kinase pathway downstream of HER2 signaling.
  • HER2+ tumors with high levels of miR- 125 and/or low levels of miR-21 would be more sensitive to herceptin treatment.
  • miR-27b and miR-206 have been implicated in the regulation of estrogen signaling; miR-206 binds and represses ERa mRNA in breast cancer cell lines (Adams, et al . (2007) Mol . Endocrinol.
  • miR-27b indirectly affects the metabolism of 17- -estradiol by repressing cytochrome P4501B1 ⁇ CYP1B1) (Tsuchiya, et al . 92006) Cancer Res. 66:9090-9098) .
  • these miRNAs could be informative for ER2+ tumors. It is believed that these miRNAs could cooperate with tamoxifen or anti -estrogenic treatments to dampen ER signaling.
  • ER+ tumors with higher levels of miR-206 and/or miR-27b would be more sensitive to anti -estrogenic treatments.
  • miRNAs such as let-7a, miR-141 and miR-221 have also been implicated in the regulation of etiological relevant proteins for .
  • breast cancer such as c-Myc (Akao, et al . (2006) Biol. Pharm. Bull. 29:903-6; Sampson, et al . (2007) Cancer Res. 67:9762- 9770), E-cadherin (Hurteau, et al . (2007) supra), and p27 (Gillies & Lorimer (2007) Cell Cycle 6:2005-2009), respectively .
  • ISH in situ hybridization
  • Table 9 provides a list of protein markers, whose codetection with specific miRNAs will be relevant for the diagnosis, prognosis, treatment, and management of breast cancer .
  • Ki-67/pl6/COX-2 and miRNAs are compared.
  • Some of these proteins have been shown to be regulated by miRNAs (e.g., in Table 8) via translational repression or relay the action of some of these proteins.
  • Clinical validation of these miRNA/protein interactions could be more informative than separate assessment of individual markers. This includes the study of E-cadherin and miR-141 as indicators of invasion and prognosis; loss of E-cadherin weakens epithelial cell-to-cell junctions and facilitates an epithelial to mesenchymal transition, which leads to cell invasion and metastasis.
  • miR-141 has been implicated in the maintenance of E-cadherin expression and thus epithelial identity via inhibition zinc-finger transcriptional factor ZEB1, which in turn repress E- cadherin gene expression (Burk, et al . (2008) supra; Gregory, et al . (2008) Nat. Cell Biol. 10:593-601; Hurteau, et al. (2007) Cancer Res . 67:7972-76).
  • codetecting miRNA expression with cytokeratins but also with other markers that specifically labeled endothelial cells (e.g., CD-31) or fibroblasts/stroma (e.g., vimentin) greatly assists in determining the cell -type distribution of miRNA expression in breast tissue and interpreting changes of miRNA in different tumor subtypes.
  • endothelial cells e.g., CD-31
  • fibroblasts/stroma e.g., vimentin
  • Additional codetections experiments include the codetection of: (a) a miRNA and protein levels of its target gene (miR-21 and PTEN, miR-125b and HER2 , miR-206 and ER, miR-221 and ER) to demonstrate the mechanistic interactions engaged in breast cancer; (b) miR-34a and P53 to demonstrate whether miR-34a could inform by proxy of p53 activity status since inactivation of p53 is an indicator of poor prognosis; (c) let-7a and Ki-67 and miR-210 and Ki- 67 to demonstrate a negative and positive correlation of miRNA expression, respectively, with cell proliferation as indicator of cell aggressiveness; and (d) miR-205 and E- cadherin and metastatic miRNAs (miR-lOb, miR-373 and miR- 520c) and E-cadherin to demonstrate a negative and positive correlation of miRNA expression, respectively, with an EMT phenotype (absent/low E-cadherin levels
  • An automated pipeline from codetection of miRNA and protein markers on high-density tissue microarrays can employ an XMATRX® staining station (BioGenex) , high-resolution image acquisition using a multi-spectral VECTRATM system (CRi) , and computer-assisted image analysis using INFORMTM software package (CRi) .
  • Major advantages of this fully automated approach include: increase experimental reproducibility, increase multiplexing capability up to eight independent markers, and much faster turn-around time from assay to data analysis.
  • XMATRX ® is an FDA-approved fully automated staining station to perform IHC, ISH and FISH assays. The XMATRX ® performs all steps from slide baking to final anti- fading media mounting and coverslipping . XMATRX ® automatically places and removes coverslips on top of the slides during critical steps, including incubations at high temperature during miRNA probe hybridization (50°C) and HIER (98°C) and incubations with small volume (30-100 ⁇ ,) to minimize cost of expensive reagents such as miRNA probes, primary antibodies and fluorochrome substrates.
  • the XMATRX ® instrument can run up to 40 different slides per experiment; each slide is placed on a separate heat block, allowing independent temperature conditions and programs. This feature is ideal for high-throughput optimization of miRNA probe conditions.
  • XMATRX ® also increases the technical reproducibility of the instant combined ISH/IHC methodology since time- and temperature-sensitive steps of Proteinase K digestion and HIER are standardized by automation .
  • VECTRATM is multi-spectral microscope capable of both fluorescence and bright field imaging.
  • VECTRATM system provides a more quantitative, more efficient and faster approach to acquire image data from whole tissue and TMA (tissue microarray) slides.
  • VECTRATM uses a unique liquid-crystal tunable system that allows the separation of fluorochromes with similar emission spectra, and also removes background autofluorescence .
  • VECTRATM codetect of up to eight signals is possible including fluorescent stains: DAPI (nuclear counterstaining for cell segmentation), AMCA, FITC, Rhodamine, Dylight594, Dylight649, and Dylight680, and chromogenic stains: DAB, BCIP/NBT and/or fast red (nuclear fast red as an alternative for counterstaining) .
  • fluorescent stains DAPI (nuclear counterstaining for cell segmentation)
  • AMCA FITC
  • Rhodamine Rhodamine
  • chromogenic stains DAB, BCIP/NBT and/or fast red (nuclear fast red as an alternative for counterstaining) .
  • the image analysis software package INFORMTM is the companion of the VECTRATM system.
  • INFORMTM identifies individual cells by detecting a nuclear marker (e.g., DAPI) and applying an algorithm for cell segmentation (essentially a nuclear and cytoplasmic outline based on nuclear size and shape, and arrangement and proximity of cells) . After cell segmentation, INFORMTM quantifies the intensity of each individual signal and keeps track of mean intensity (and other parameters) for each defined cell.
  • a nuclear marker e.g., DAPI
  • a cell type-specific marker e.g., CK19 for epithelial/cancer cells
  • CK19 for epithelial/cancer cells
  • a similar analysis can be performed to assess regulatory interactions in clinical specimens between specific miRNAs and their target genes.
  • image capturing and analyzing platforms such as ScanScope FL system and accompanying ImageScope software package by Aperio, Pannoramic Fluorescent Scanner by 3DHistech, and VS110 for Fluorescence by Olympus, and custom-designable MicroVigene software package by VigeneTech that are amenable and programmable to perform data acquisition and analysis of this present multi-color combined ISH/IHC assay .
  • cancer-associated miRNAs refers to those miRNAs whose levels change between normal and tumor tissue in carcinomas of the breast, colon, lung and/or pancreas and those miRNAs which have been shown to exhibit oncogenic or tumor suppressive in cell line systems and/or mouse models of these solid tumors. miRNAs can be grouped into broad categories with respect to their reported tumor suppressive and oncogenic functions.
  • the tumor suppressive group includes those miRNAs with anti -proliferative , pro- apoptotic and/or anti-metastatic properties: let-7s, miR- 15, miR-16, miR-26b, miR-27b, miR-34s, miR-125b, miR-141, miR-200s, miR-205, and miR-335.
  • the oncogenic group includes those miRNAs with pro-mitogenic , anti -apoptotic and/or pro-metastatic properties: miR-lOb, miR-17, miR-18, miR-19, miR-20, miR-21, miR-23s, miR-31, miR-106b, miR-206, miR-214, miR-221, miR-222, miR-373, and miR-520c.
  • miR-lOb miR-17, miR-18, miR-19, miR-20, miR-21, miR-23s, miR-31, miR-106b, miR-206, miR-214, miR-221, miR-222, miR-373, and miR-520c.
  • Other cancer-associated miRNAs categorized by changes in RNA levels in tumor tissues are listed in Table 10.
  • miRNAs detected at lower levels in tumor tissue miR-126, miR-143, and miR-145 and miRNAs detected at higher levels in tumor tissue: miR-107, miR-132, miR-155, miR- 196s, miR-210, and miR-213.
  • miR-29 family miR-142, miR-146, and miR-181.
  • ISH detection assay denotes exclusive expression
  • expression profiling assays indicates that changes were observed in tumor-associated fibroblast or tumor-associated vasculature.
  • Arrows indicate general trend of increase ( ⁇ ) or decrease ( ) miRNA levels in whole tumor tissue specimens from breast, colon, lung, and pancreas.
  • ISH/IHC assay can use three or four fluorescent stains for detection of miRNA, U6 snRNA, cell type protein markers and other protein markers, up to three more protein markers can be codetected.
  • These proteins include the product of target genes, regulators of miRNA expression and proteins that reflect a biological output downstream of the miRNA- regulated event (s) (Table 11).
  • miR-34 family members miR-34a,b, c
  • miR-34s are transcriptionally activated by the p53 tumor suppressor gene in response to DNA damage and mitogenic signals. Consistent with a tumor suppressive role, miR-34s were detected at lower levels in breast, lung and other solid tumors by expression profiling analyses in whole tissue biopsies.
  • Codetection of these miRNAs and p53 may identify different molecular subtypes of functional, null or mutated p53.
  • expression of miR-34a or other miRNAs could inform by proxy of p53 activity status and provide a better prognostic indicator than a p53 -based IHC assay alone .
  • miR-21 is expressed at high levels within cancer cells (lung and pancreatic cancer) and tumor- associated fibroblasts (TAFs) (breast and colorectal cancer) compared to matched normal tissues (Sempere, et al . (2007) supra) , as supported by co- staining with the mesenchymal markers vimentin and smooth muscle actin.
  • TAFs tumor-associated fibroblasts
  • ISH assay results generated using LNA-modified probes against candidate tumor-suppressive or oncogenic miRNAs . These assays were performed using formalin- fixed, paraffin- embedded normal or malignant lung tissues. The results showed the miRNA expression patterns of the indicated miRNAs in age-matched, nontransgenic sibling controls (normal lung tissue) and in a cyclin E-driven mouse model of lung cancer.
  • ISH expression patterns for miR-31 and for the 18S ribosomal RNA (as control for RNA integrity) in paired human normal lung tissue versus adjacent lung cancer (adenocarcinoma) were generated. This ISH analysis confirmed augmented miR-31 expression (relative to adjacent normal lung) within both murine and human lung cancers. Representative hematoxylin and eosin stained lung tissues were also prepared.
  • Clinical specimens were placed on a slide. The slide was heated to 65°C for 30 minutes and subsequently allowed to cool to 26°C for 1 minute. The sample was dewaxed for 5 minutes with xylene and the slide was washed 2X 30 seconds with alcohol (ethanol) , IX 30 seconds with DI water, and 3X 30 seconds with IHC wash. Proteinase K (50 ⁇ ,; 10 pg/ml) was applied to the sample, and the sample was incubated at 37°C for 20 minutes. The sample was washed for 20 seconds with xylene. The sample was subsequently incubated in a solution of 0.2% glycine (in PBS; 300 pL) for 1 minute and washed 3X 30 seconds with IHC wash.
  • 0.2% glycine in PBS; 300 pL
  • Probe Hybridization Prehybrization solution (50 pL) was added to sample and the sample was incubated at 45°C for 15 minutes. Double FAM-tagged (5' and 3' ends) miR-155 probe (50 L) was added to the sample, and the sample was incubated at 50°C for 75 minutes. The sample was washed for 30 seconds in ISH wash and subsequently washed in SSC buffer for 5 minutes at 50°C, DEPC water for 30 seconds, ISH wash for 30 seconds, and DEPC water for 30 seconds. Hydrogen peroxide (300 pL) was added to the sample for 15 minutes to block endogenous horseradish peroxidase activity. The sample was again washed with ISH wash for 30 seconds, DEPC water for 30 seconds, and ISH wash for 30 seconds. The sample was subsequently blocked with BSA (300 pL) for 20 minutes followed by a 5-minute was in PBT .
  • BSA 300 pL
  • Anti-FITC primary antibody 50 pL was incubated with the sample for 45 minutes at 37°C, and the sample was subsequently washed for 2 minutes each with DEPC water, ISH wash, and DEPC water. Subsequently, an anti-rabbit horseradish peroxidase (HRP) - conjugated antibody (50 ]iL; 1:500 dilution) was incubated with the sample for 30 minutes at 37°C followed by consecutive washes in DEPC water (2 minutes) , ISH wash (2 minutes) , and DEPC water (2 minutes) .
  • HRP horseradish peroxidase
  • TSA green substrate 50 ⁇ ; 1:200 dilution
  • TSA green substrate 50 ⁇ ; 1:200 dilution
  • the sample was incubated for 20 minutes at 25 °C.
  • the sample was subsequently washed in ISH wash for 2 minutes, DEPC water for 2 minutes, and ISH wash for 2 minutes.
  • Hydrogen peroxide 300 ⁇ was added to the sample for 15 minutes and the sample was washed consecutively for 30 seconds in each of DEPC water, ISH wash, and DEPC water.
  • PBT 300 ⁇ was added to the sample for 5 minutes followed by a 45 minute incubation in streptavidin-conjugated HRP (50 ⁇ il>) at 37 °C.
  • the sample was washed for 2 minutes in each of ISH wash, DEPC water, and ISH wash and TSA red substrate (50 L; 1:500 dilution) was added for 15 minutes at 25°C.
  • the sample was again washed for 2 minutes in DEPC water, 2 minutes in ISH wash and 2 minutes in DEPC water.
  • the sample was subjected to HEIR at 90°C for 10 minutes and washed briefly and consecutively in ISH wash, DEPC water, and ISH wash.
  • Hydrogen peroxide 300 ⁇
  • the sample was incubated with BSA (300 ⁇ for 20 minutes and washed with PBT for 5 minutes.
  • Primary anti-CD68 IgGl antibody 50 pL was incubated with the sample for 30 minutes at 37°C. Subsequently, the sample was washed for 2 minutes in DEPC water, 2 minutes in ISH wash, and 2 minutes in DEPC water. Secondary ant i- IgGl isotype-specific HRP-conjugated antibody was added to the sample and the sample was incubated at 37°C for 30 minutes. The sample was briefly washed in ISH wash and DEPC water.
  • TSA blue substrate 50 ⁇ ,; 1:500 dilution
  • Hydrogen peroxide 300 ⁇ , was added to the sample for 15 minutes and the sample was washed consecutively for 30 seconds in each of ISH wash, DEPC water, and ISH wash.
  • the sample was washed with PBT for 5 minutes and anti-CD3 IgG2 antibody (50 ⁇ ) was added to the sample for 30 minutes at 37°C.
  • the sample was washed for 2 minutes in DEPC water, 2 minutes in ISH wash, and 2 minutes in DEPC water.
  • Secondary anti-IgG2a isotype- specif ic antibody 50 ⁇ was incubated with the sample for 30 minutes at 37°C, and the sample was subsequently washed for 20 seconds in each of ISH wash, DEPC water, and ISH wash.
  • TSA daylight 594 substrate 50 ⁇ . ; 1:100 dilution
  • Hydrogen peroxide 300 ⁇ .
  • the sample was washed with PBT for 5 minutes and anti- PO rabbit antibody (50 ⁇ ) was incubated with the sample for 30 minutes at 37°C.
  • Example 16 Protocol for Combination of Chromogenic and Fluorescent Staining to Codetect Protein and miRNA Markers in Cancer Tissue Specimens
  • Clinical specimens were placed on a slide.
  • the slide was heated to 65°C for 30 minutes and subsequently- allowed to cool to 26°C for 1 minute.
  • the sample was dewaxed for 5 minutes with xylene and the slide was washed 2X 30 seconds with alcohol (ethanol) , IX 30 seconds with DI water, and 3X 30 seconds with IHC wash.
  • Proteinase K 50iL; 10 g/ml was applied to the sample, and the sample was incubated at 37 °C for 20 minutes.
  • the sample was washed for 20 seconds with xylene.
  • the sample was subsequently incubated in a solution of 0.2% glycine (in PBS; 300 ⁇ ,) for 1 minute and washed 3X 30 seconds with IHC wash.
  • Probe Hybridization Prehybrization solution (50 ]iL) was added to sample and the sample was incubated at 45°C for 15 minutes. Labeled miR-21 probe (50 pL) was added to the sample, and the sample was incubated at 45°C for 75 minutes. The sample was washed for 30 seconds in ISH wash and subsequently washed in SSC buffer for 5 minutes at 45°C, DEPC water for 30 seconds, ISH wash for 30 seconds, and DEPC water for 30 seconds. Hydrogen peroxide (300 i ) was added to the sample for 15 minutes to block endogenous horseradish peroxidase activity. The sample was again washed with ISH wash for 30 seconds, DEPC water for 30 seconds, and ISH wash for 30 seconds. The sample was subsequently blocked with BSA (300 yL) for 20 minutes followed by a 5-minute was in PBT.
  • BSA 300 yL
  • Antibody Binding and TSA Anti-FITC primary antibody (50 yL) was incubated with the sample for 45 minutes at 37°C, and the sample was subsequently washed for 2 minutes each with DEPC water, ISH wash, and DEPC water. Subsequently, an anti-rabbit horseradish peroxidase (HRP) - conjugated antibody (50 il>; 1:500 dilution) was incubated with the sample for 30 minutes at 37°C followed by consecutive washes in DEPC water (2 minutes) , ISH wash (2 minutes) , and DEPC water (2 minutes) . TSA green substrate (50 yL; 1:200 dilution) was added and the sample was incubated for 20 minutes at 25°C.
  • HRP horseradish peroxidase
  • ISH wash was subsequently washed in ISH wash for 2 minutes, DEPC water for 2 minutes, and ISH wash for 2 minutes.
  • Hydrogen peroxide 300 yL was added to the sample for 15 minutes and the sample was washed consecutively for 30 seconds in each of DEPC water, ISH wash, and DEPC water.
  • PBT 300 yL was added to the sample for 5 minutes followed by a 45 minute incubation in streptavidin-conjugated HRP (50 yL) at 37 °C.
  • the sample was washed for 2 minutes in each of ISH wash, DEPC water, and ISH wash and TSA red substrate (50 yL; 1:500 dilution) was added for 15 minutes at 25°C.
  • the sample was again washed for 2 minutes in DEPC water, 2 minutes in ISH wash and 2 minutes in DEPC water.
  • Hydrogen peroxide 300 ⁇ was again added to the sample for 15 minutes and the sample was washed consecutively for 30 seconds in each of ISH wash, DEPC water, and ISH wash.
  • the sample was washed with PBT for 5 minutes.
  • Primary anti-CK19 antibody 50 yL; 1:200 was incubated with the sample for 30 minutes at 37°C. Subsequently, the sample was washed for 2 minutes in DEPC water, 2 minutes in ISH wash, and 2 minutes in DEPC water.
  • Hydrogen peroxide 300 ih was again added to the sample for 15 minutes and the sample was washed consecutively for 30 seconds in each of ISH wash, DEPC water, and ISH wash.
  • the sample was incubated with BSA (300 ⁇ ]) for 20 minutes.
  • the sample was washed with PBT for 5 minutes and anti-Her2 mouse antibody and anti-Pten antibody (50 ⁇ _) were added to the sample for 30 minutes at 37°C.
  • the sample was washed for 2 minutes in DEPC water, 2 minutes in ISH wash, and 2 minutes in DEPC water.

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Abstract

La présente invention concerne des méthodes et un kit de codétection de micro-ARN et de protéines dans des échantillons biologiques, tels que des spécimens fixés au formol et inclus en paraffine, par un dosage combiné immunohistochimique et d'hybridation in situ.
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EP3436600B1 (fr) * 2016-03-30 2024-01-17 Genfit Diagnostic non invasif de la stéatose hépatique non alcoolique

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